Identifying Intrinsic Regulators Of Areal Patterning In The Neocortex by Single-Cell RNAseq And Organoid Arealization Project Summary/Abstract The mature human neocortex (Ncx) is an incredibly complex structure, which is organized into functionally specialized areas, unique in their cytoarchitecture and connectivity patterns to the rest of the brain. Cortical areas are selectively dedicated to distinct processes, ranging from cognition and sensory perception to motor output. All neocortical excitatory neurons (ExNs) arise from a common progenitor pool of radial glia (RG), the neural stem cells of the Ncx, and their transit-amplifying intermediate progenitor cells (IPCs). Newborn ExNs then migrate to the cortical plate along the RG scaffold, in a process conserved across prospective areas. However, the mature Ncx is a mosaic of ExN populations. It is currently accepted that Ncx arealization is the result of a combination of intrinsic genetic mechanisms, including transcription factors (TFs) differentially expressed by progenitors, and of extrinsic influences, primarily in the form of thalamocortical input. The relative contribution of these two components, however, is not well understood. Over the past 30 years, seminal studies have uncovered molecular differences across cortical areas, as well as genes crucial for areal specification, but still there remains much to be explained. Recent advances in single-cell sequencing have begun to illuminate additional cell type diversity that exists in mouse and human brains, with significant transcriptional areal differences between otherwise corresponding ExN subtypes. Understanding how these differences emerge is essential to understanding how neurodevelopmental disorders may arise, as well as to more accurately model human cortical cell types and understand how stem cell therapies may best be developed in an area specific manner. A systematic characterization of RG and ExNs across areas of the developing Ncx is a powerful approach to study intrinsic arealization factors. To this end, we have previously generated a spatially-annotated dataset of ~550,000 single-cell transcriptome profiles from distinct areal subdissections of the Ncx during the period of peak neurogenesis. My analysis will focus on identifying molecular subtypes of RG and ExNs specific to prospective areas of the developing Ncx, and determining their unique genetic markers and expression signatures, with a particular emphasis on TFs. Using cortical organoids, I will evaluate the capacity previously described key arealization genes to drive spatial identity in a human context. Building on this system, I will evaluate the functional importance for arealization of 2 TFs identified in my preliminary analyses as being preferentially expressed by RG of frontal and occipital Ncx, respectively. Through this work, I aim to further shed light on the intrinsic mechanisms that shape diversity across areas of the human neocortex.